The general aim of this project is to elucidate mechanisms by which proteins and viruses enter cells by receptor-mediated transport processes and translocate across the lipid bilayer to cytosol and nuclear compartments. Molecular signals which lead to unique vesicle trafficking are studied. Basic knowledge from these processes is used to design targeted drug delivery systems such as immunotoxins. Anti-human CD3 immunotoxins constructed with CRM9, a binding site mutant of diphtheria toxin, eliminate 50% of established tumors of human T cell leukemia in a nude mouse xenograft system. The therapeutic margin appears promising for clinical application which might include treatments for &n, autoimmune diseases and graft-versus-host-disease following bone marrow transplantation. Differential routing of receptors following endocytosis has been found to be a major variable affecting immunotoxin efficacy. CM and CD3 are both rapidly endocytosed and recycled back to the plasma membrane, yet only the anti-CD3 immunotoxin intersects the diphtheria toxin pathway and is toxic. Immunotoxins are effective agents for the selection of endocytotic routing mutants. A series of Thy1 routing mutants have been isolated and cloned following selection with anti-Thy1 diphtheria toxin. The routing mutants are Thy1 positive, sensitive to toxin but resistant to the immunotoxin. A (bis)succinimidyl protein crosslinking agent has been synthesized which contains an acid-labile ketal bond. This permits easy identification of protein species previously crosslinked in situ by exposure to mild acid prior to gel electrophoresis.